The overall objective of the project is a fundamental understanding of the biologically important chemistry of lipid-derived endoperoxides and reactive products generated from these key intermediates in oxidative transformations of polyunsaturated fatty acids. The project focuses on identifying pathological modifications of proteins and other biomolecules resulting from reactions with lipid oxidation products. The conversion of low density lipoprotein (LDL) into an atherogenic oxidatively damaged form (oxLDL) is a pertinent example. Although oxLDL is known to be associated with the development of atherosclerotic plaques, little was known about the molecular structures of the lipid- derived modifications present in oxLDL. We previously identified some of these structures as protein-bound pyrrole derivatives of levuglandins (LGs). Using total synthesis and immunological techniques, during the previous funding period we demonstrated that: 1) a family of structural isomers of LGs (isoLGs) and a family of carboxyalkylpyrrole (CAP) protein modifications are produced by free-radical oxidative processes; 2) LG- and isoLG-protein adducts as well as CAPs are present in atherosclerotic plaques, oxLDL, and human blood and levels are significantly elevated in atherosclerosis and renal failure patients compared with healthy individuals; and 3) LGE2-LDL is recognized and taken up by macrophage cells. These developments provide new opportunities to obtain a molecular level understanding of the biological consequences of lipid oxidation. Thus, levels of specific lipid oxidation products can provide information about the involvement of enzymatic versus free radical pathways and of particular polyunsaturated fatty acids in disease-specific oxidative injuries. Over the next four years we will also test hypotheses: 1) that a family of nLGs is generated through free radical oxidation of docosahexaenoic acid-based lipids; 2) that CAPs are produced from gamma-hydroxy- alpha,beta-unsaturated aldehyde derivatives of lipid esters, "core aldehydes", with chemical reactivity analogous to 4-hydroxy-2-nonenal, a well-known cytotoxic lipid oxidation product; 2) that these core aldehydes are biologically active, e.g. inhibit enzymes; 3) that specific lipid-derived protein epitopes are biologically active, e.g. induce uptake by macrophages, enhance an "oxidative burst" in vascular cells, or trigger the apoptotic response to photodynamic treatment; 4) that modification of ethanolamine phospholipids by pyrrole-forming reactions with lipid oxidation products converts cationic ammonium groups into anionic carboxylate derivatives resulting in ejection from the outer shell of LDLs and causing pathological alterations in membranes.